World Journal of Pharmaceutical Research et al …KEYWORDS: Karanj oil, Pongamia Pinnata, UPLC,...

www.wjpr.net Vol 6, Issue 17, 2017.

846

Nagaiah et al. World Journal of Pharmaceutical Research

METHOD VALIDATION AND SIMULTANEOUS DETERMINATION

OF TWO BIO-ACTIVE MARKER COMPOUNDS IN PONGAMIA

PINNATA: SEED AND KARANJ OIL BY UPLC-MS

N.M.A. Rasheed1, K. Nagaiah

1*, G. Sowmya Srividya

1 and M.V.N. Kumar Talluri

2

1Organic & Biomolecular Chemistry, CSIR-Indian Institute of Chemical Technology,

Tarnaka, Hyderabad-500007.

2National Institute of Pharmaceutical Education and Research (NIPER), Balanagar,

Hyderabad, 500037.

ABSTRACT

Seed of Pongamia pinnata (Linn.) Merr known as "Karanj" widely

used in traditional system of medicine. Oil from karanj seeds contain

flavonoids which possess diverse pharmacological properties. The seed

oil was subjected to karanj oil-liquid extraction with methanol to

isolate karanjin and pongamol which are active principle of karanj. The

oil mainly used for skin diseases such as psoriasis, eczema and vitiligo

etc. A validated reversed-phase ultra-performance liquid

chromatography method with photodiode array detector was

successfully developed for the first time report to simultaneously

determine two active compounds karanjin and pongamol in karanj seed

extract and its oil. Separation carried through a BEH C18 column by

gradient elution using 0.1% formic acid in water (v/v): methanol:

acetonitrile at 0.2 mL/min. Significant linear correlations were found between component

concentrations and specific chromatographic peak areas. The % R.S.D. values found by the

method indicating precision, range and recovery to be good. Thus the proposed validated

method can be applied as a reference standard for quality control analysis commercial

samples of karanj oil.

KEYWORDS: Karanj oil, Pongamia Pinnata, UPLC, Karanjin, Pongamol.

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 6, Issue 17, 846-857. Research Article ISSN 2277–7105

*Corresponding Author

Dr. K. Nagaiah

Senior Principal Scientist,

Organic & Biomolecular

Chemistry, CSIR-Indian

Institute of Chemical

Technology, Tarnaka,

Hyderabad-500007.

Article Received on

22 Oct. 2017,

Revised on 13 Nov. 2017,

Accepted on 04 Dec. 2017,

DOI: 10.20959/wjpr201717-10395


847


INTRODUCTION

The seeds of Pongamia pinnata (Linn.) Merr, syn. Pongamia glabra vent. (Family-

Leguminosae) mostly used in traditional Indian system of medicine particularly in Unani and

Ayurveda and popularly known as karanj in urdu, karanja in hindi. It is a medium sized

glabrous tree spread throughout India at an altitude of 1200 m. Macroscopically seed of

karanj appear to be one and rarely two elliptic or somewhat reniform in shape, testa as

reddish leathery. Transverse section of karanj seed shows testa as composed of a layer of

palisade like outer epidermis, filled with brown pigment, covered externally with a thick

cuticle, thick-walled parenchyma cells of two to four layers, and a number of parenchyma

cells containing brown pigment.[1]

All parts of P. pinnata are therapeutically useful for

treating tumors, piles, skin diseases, wounds and ulcers.[2]

The names and structure of the

chemical principles isolated from P. pinnata is given in Fig-1.

Karanjin

Pongamol

Pongarotene

Kanjone

Pongagalabrone

Pongapin

Pinnatin

Pongone

Glabrachalcone

Glabone

Fig. 1: Chemical structures of constituents of P. Pinnata L.[3-5]


848


Karanj oil from seeds contain two important compounds karanjin and pongamol, both are

bioactive compounds with important biological attributes. Seed reported to possess

anthelmintic activity, effective in treatment of leprosy, chronic fever, also helps in piles,

relives liver pain, heals ulcers.[6]

Seed extracts possesses antiinflammatory activity,[7]

anti-

oxidative, anti-ulcerogenic properties, analgesic and hypoglycaemic,[8]

also useful in

rheumatism arthritis and scabies[9]

and it is used to treat dermatitis of pet animals[10-11]

and

gastroprotective.[12]

Various herbal remedies separately or along with combination were

recommended in different medical conditions for the cure of skin diseases such as psoriasis,

eczema and vitiligo.[13]

The oil reported to be antihelmintic, styptic and recommended for

opthalmia, leprosy, ulcers, herpes and lumbago. Its oil utilized as a source of biodiesel.[14-15]

Karanj oil reported to contains 5–6% flavonoids, the main active constituents found are

karanjin, (a furano-flavonoid) and pongamol (a diketone) which possess various

pharmacological properties[16]

and their isolation in pure form and efficient quantitative

method with more precise and accurate content in seed extracts, seed oil and also

commercially available oil samples at present a great need.[17]

Karanjin showed pesticidal and

insecticidal properties.[18]

Dermal absorption of karanjin from Jatyadi Taila an oil based

Ayurvedic drug.[19]

The presence of karanjin, pongamol, pongagalabrone, pongapine,

pinnatine and kanjone in Pongamia pinnata which are mainly responsible for various

pharmacological activity.[20]

(Fig-1)

Another use of karanjin oil as starting material for biodiesel, the manufactures do not seem to

remove karanjin, pongamol etc for biodiesel which can be more useful for medicinal purpose

as described above. Karanj oil also used to light lamps, where karanjin, pongamol and other

compounds and other active principles are not required. Biodiesel is prepared by trans-

esterification of karanj oil by heating with NaOH or KOH and methanol. Karanjin is not

desirable in biodiesel because it corrodes the engine parts. If karanjin is removed before, the

biodiesel is efficient. On the other hand, the crude karanjin and pongamol enriched oil as

shown in the present study, could be used in skin ointments.

Earlier few methods were reported by Gore and Satyamoorthy, Shejawal et al.[11,21]

which are

based on conventional HPLC for karanjin only and for both compounds based on dual

wavelength detection and are not validated. The objective of this study was to develop a

validated UPLC method for simultaneous determination of karanjin and pongamol as per ICH

guidelines[22]

at a single wavelength. The seed oil was subjected to karanj oil–liquid


849


extraction with methanol to isolate pure compounds of karanjin and pongamol[13]

and

preparation of enriched oil for topical application in the treatment of skin diseases. Till now

no method was reported through UPLC for the estimation of karanjin and pongamol in

commercial or marketed samples to the best of our knowledge. Thus efforts were made to

develop a validated RP-UPLC method by PDA.

MATERIAL AND METHODS

All HPLC grade reagents were used. HPLC-grade methanol, acetonitrile, water, formic acid,

orthophosphoric acid, triflouroacetic acid were used. Marketed Samples of karanj oil such as

Vyas karanj oil and Cold pressed pure karanj oil make Soul-centric was procured and karanj

seeds were procured from the pharmacy, Central Research Institute of Unani Medicine,

Hyderabad and one from local region. Karanjin and pongamol (figure 1) are the pure isolated

compounds from Pongamia pinnata and characterized through spectrophotometric methods

and used as reference standards. These pure compounds available in our laboratory. Karanj

oil is enriched with respect to karanjin etc., by oil/liquid extraction with methanol or

incorporating technical karanjin into oil. Syringe filter PTFE membrane of 0.22 µm pore

size, dia. 25mm (Axiva).

Standard preparation

Standard stock solutions of karanjin and pongamol were made with 10mg in 10ml with

methanol to obtain 1000 μg/ml each separately. Aliquots of stock solutions were prepared by

dilution to obtain required concentrations of 10, 20, 30, 40, 50 g/ml and 2l was injected.

Sample preparation

Samples of karanj oil and seeds were accurately weighed one gram each and transferred in

100 ml beaker, and extracted with 10ml methanol by ultrasonic extraction method for 15 min.

The sample extracts filtered through syringe filter PTFE membrane of 0.22 µm pore size,

stored and used for analysis.

Chromatographic conditions

The mobile phase before delivering was filtered through 0.22 m, PTFE filter and sonicated

with the help of ultrasonicator for 15 minutes. The analysis was carried out under gradient

conditions at a flow rate of 0.2μL/min at temperature 40oC and recorded at 350nm

wavelength.


850


Instrument conditions

Acquity UPLC-H class, a quaternary pump, and PDA detector was used. Analyses were

carried on Acquity BEH C18 reversed-phase analytical column (2.1 × 100 mm, 1.7 μm), flow

rate of 0.2 mL/min, at 350 nm wavelength, injection volume 2 μL, column temperature 40°C.

The mobile phase A (0.1% formic acid in water), solvent B (methanol) and solvent C

(acetonitrile) filtered through a 0.22 μm membrane filter; gradient elution as follows: 0 min

40% A, 40% B, 20%C; 1 min 37% A, 48% B, 15%C; 8 min 34% A, 56% B, 10%C; 9 min

30% A, 60% B, 10%C; 11 min 25% A, 65% B, 10%C; 15 min 40% A, 40% B, 20%C; 18

min 40% A, 40% B, 20%C. The data were collected and analyzed using Waters Software

Empower 3.

RESULTS AND DISCUSSIONS

Method development and optimization

Using isocratic elution, all the compounds cannot be effectively separated; thus, gradient

elution was used throughout the study. Suitable wavelength selected for simultaneous

determination of karanjin and pongamol in the range of 200-400 nm and both had good

absorbance at 350 nm. The chromatographic variables were also optimized, including column

temperatures and column types. Optimum separation was eventually achieved with the

reversed-phase Waters Acquity UPLC™ BEH C18 2.1 x 100 mm, 1.7 µm, at 40°C

temperature. To obtain chromatograms with well-resolved peaks and comfortable analysis

time/run, chromatographic conditions such as column temperature, type of reversed-phase

column, extraction solvent, detection wavelength, and mobile phase along with buffer were

optimized. In this study, methanol, acetonitrile and water along with a small amount of

formic acid as buffer was added to the mobile phase. The acid inhibited the ionization of

acidic compounds in the samples to improve peak shape and restrain peak tailing.

Subsequently 0.1% formic acid in the mobile phase significantly improved the retention

behavior and peak shape of the different components in karanj oil samples. The following

optimized chromatographic conditions were obtained for analysis as mobile phase A: (0.1%

FA in water) and B: (methanol) and C: (acetonitrile). Gradient conditions for elution as 0 min

40% A, 40% B, 20%C; 1 min 37% A, 48% B, 15%C; 8 min 34% A, 56% B, 10%C; 9 min

30% A, 60% B, 10%C; 11 min 25% A, 65% B, 10%C; 15 min 40% A, 40% B, 20%C; 18

min 40% A, 40% B, 20%C. The flow rate was 0.2mL/min, and the column temperature was

40°C. A typical UPLC chromatogram as shown in figure 2. The retention times for the

compounds detected in the Karanj oil samples were at karanjin (tR 7.3 min) and pongamol (tR


851


13.6 min). Upon LCMS analysis, karanjin and pongamol was detected in the positive mode

were identified and confirmed in standard solution and in the Karanj oil samples through

LCMS data.

Peak1 K

ara

njin

- 7

.388

Peak2 P

ongam

ol -

13.6

48

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Fig. 2. UPLC chromatogram of (a) standard mixture, b) UV Spectrum of karanjin and

pongamol of standard and sample c) karanj oil sample.

a: std mix

Karanj oil sample

7.393 Peak9 Karanjin

217.3

260.1

306.3

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

13.642 Peak12 Pongamol

235.6

350.3

AU

0.000

0.010

0.020

0.030

0.040

nm

220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

7.387 Peak1 Karanjin

217.3

260.1

306.3

AU

0.00

0.20

0.40

0.60

13.648 Peak2 Pongamol

239.3

349.1

AU

0.000

0.010

0.020

0.030

0.040

0.050

nm

220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

Peak1 -

2.5

43

Peak2 -

4.0

51

Peak3 -

4.3

51

Peak4 -

5.1

24

Peak5 -

5.7

08

Peak6 -

6.0

74

Peak7 -

6.6

18

Peak8 -

7.0

00 Peak9 K

ara

njin

- 7

.393

Peak10 -

8.0

83

Peak11 -

13.2

13

Peak12 P

ongam

ol -

13.6

42

Peak13 -

14.0

86AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

b: sample UV spectrum b: std UV spectrum

c:


852


System suitability

To ensure adequate performance of the chromatographic system, we evaluated the retention

time (tR), Resolution (RS), number of theoretical plates (N), and tailing factor (T) using

standard solution for karanjin and pongamol such as retention time (tR), Resolution (RS),

number of theoretical plates (N), and tailing factor (T), all parameters were good and in

acceptable limits.

Linearity, limit of detection and limit of quantitation

After establishing the optimum conditions, method validation was performed. Good linear

correlation and high-sensitivity under these chromatographic conditions were confirmed by

the correlation coefficients (R2), limits of detection (LOD), and limits of quantitation (LOQ)

The linearity calibration curves were plotted based on at least five calibration points

performed in triplicate. The correlation coefficient R2 and linear regression equations were

analyzed. Results of regression analyses and the calculated correlation coefficients (R2)

indicated good linearity within concentration ranges for both the investigated compounds at

the wavelengths at 350 nm as given in table 1.

Table 1: Statistical results of linear regression equation analysis in the determination of

the two investigated compounds.

S.no Parameter Karanjin Pongamol

1 Linear Range (μg/ml) 10-50 10-50

2 Regression equation y = 96615x - 699.7 y = 93440x - 64619

3 R2 0.998 0.995

Limits of detection and limits of quantitation under the present chromatographic conditions

were defined as the lowest concentration with the signal-to-noise ratio of 3 and 10 as criteria,

respectively. The LODs for karanjin and pongamol was 0.25 and 1.41 μg/ml respectively and

LOQs for karanjin and pongamol was 0.86 and 4.70 μg/ml respectively.

Precision, repeatability and recovery data

Precision was evaluated by analyzing standard samples. Thus, five individual sample

solutions were analyzed. For each compound the RSD of the mean was calculated and ranged

from 0.13% to 0.60% for intraday precisions. Hence the proposed method was found to be

precise. The results are shown in table 2. The recoveries of karanjin and pongamol were

assessed by spiking known amount each at three different levels ranging 50µg, 100 µg and

150 µg with respect to concentration of karanjin and pongamol in karanj oil samples found


853


and further analyzed by UPLC. All estimations were repeated thrice (n=3) and the relative

standard deviations were calculated. The recovery range and %RSD for karanjin and

pongamol were found to be 96.94-99.32% and 100.55-105.25% with %RSD < 0.0018%

respectively as showed in table 2.

Table 2: Precision, Repeatability and Recovery of two compounds.

Karanjin Pongamol

Repeatability

(Mean ± %RSD)

(n=3)

11873382± 0.16 14939121± 0.13

Recovery

(Mean ±%RSD)

(n=3)

99.32 ±0.0018 100.55 ±0.0013

Precision Karanjin Precision Pongamol

Conc.

𝛍g/ml

Intraday (n=3) Inter day

(n=3) Conc.

𝛍g/ml

Intraday (n=3) Inter day

(n=3)

Mean ± %RSD Mean

± %RSD

Mean

± %RSD

Mean

± %RSD

10 97913 ± 0.53 97166 ± 0.86 10 97913 ± 0.53 97166 ± 0.86

20 191663 ± 0.13 192235 ± 0.99 20 191663 ± 0.13 192235 ± 0.99

30 290404 ± 0.28 292826 ± 0.58 30 290404 ± 0.28 292826 ± 0.58

40 377862 ± 0.10 381031 ± 0.65 40 377862 ± 0.10 381031 ± 0.65

50 487890 ± 0.33 494916 ± 1.38 50 487890 ± 0.33 494916 ± 1.38

Similarly the accuracy in determination of the assay of karanj oil sample was checked at five

concentration levels i.e. 10, 20, 30, 40, 50µg/ml each in triplicate for 3 days and the

percentage recoveries are recorded in table-2. % RSDs were calculated to measure the

method reproducibility. Results indicated that the RSDs of all detected compounds were

<1.5%, which indicated that the develop method had good reproducibility. The accuracy of

the method was found to be good with the overall % RSD for recovery at known amount

addition of 50µg, 100 µg and 150 µg levels were all within the limits. This indicates that the

proposed method was found to be accurate.

Karanj oil Sample analysis

The assay of six different samples of P. pinnata and enriched karanj oil was subjected to

methanolic extraction through ultrasonication method separately. Extract was filtered through

0.22μm PTFE syringe filter; thus the solution obtained was diluted to 10 times and used for

analysis. Under the optimized chromatographic conditions the results obtained are

represented in table-3. The corresponding overlay chromatogram of formulations with

separation of peaks in seven batches as shown in figure-3. Results indicated that the UPLC


854


method had good precision, repeatability and recovery as shown above and that the

developed assay was reliable and useful for assessing Karanj oil quality. The developed

UPLC-PDA method was also reproducible, highly precise, and thus satisfactory for

quantitative analysis.

Table 3. The content of karanjin and pongamol in karanj oil samples.

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Fig. 3. Overlay chromatogram of (1-7) samples of karanj oil.

Sample

code

Sample

name

karanjin pongamol

Retention

time Area

karanjin

content

found

% w/w

Retention

time Area

pongamol

content

found

% w/w

1 AKO Market

karanj oil 7.396 1092110 1.11 13.635 1289441 0.15

2 CPKO

Cold

pressed

karanj oil

7.377 565254 0.57 13.627 1002196 0.11

3 VKO Vyas karanj

oil 7.376 419464 0.43 13.63 511044 0.06

4 KNGS Local seed

samples 7.388 1072271 1.09 13.629 978765 0.11

5PH1

Pharmacy

seeds

sample 1

7.393 1921373 1.95 13.626 2390067 0.27

6 PH2

Pharmacy

seeds

sample 2

7.391 1561014 1.59 13.628 1360519 0.16

7 KEE Karanj

enriched oil 7.391 3409626 3.46 13.639 3964015 4.53


855


The amount of karanjin and pongamol in karanj oil were found in the range 0.43-1.95 %w/w

and 0.06-0.27%w/w respectively (table-3). The karanjin and pongamol content in the

enriched karanj oil was 3.46%w/w and 4.53 %w/w respectively (table-3, 7KEE).

CONCLUSION

Karanj oil possess antibacterial, antifungal properties for which it is used externally or

topically on skin to treat or heal fungal skin diseases like itching and eczema etc. Karanj oil

whether marketed or cold pressed and karanj seeds from pharmacy or from local region

which were used in the study shows slight variation in their content of karanjin and pongamol

assessed as shown above. Karanj oil sold commercially be used for medicinal purpose should

contain 3-4 percent of karanjin and pongamol active principle for the efficacy. Thus suitable

skin ointment formulations of karanj oil if made with enriched oil so as to have 3-4 % of

karanjin and pongamol may have high potency. If pure karanjin is used in ointment that

becomes an allopathic ointment which are not suitable to use topically and also to make unani

or ayurvedic based herbal ointment. The study also describes a reversed-phase UPLC-PDA

method was successfully developed and validated following ICH guidelines for the

simultaneous detection of two active marker compounds the karanjin and pongamol in karanj

oil. The study serves as a reference standard for the quality control check for karanj oil

analysis.

ACKNOWLEDGMENT

The author thanks Director, CSIR-Indian Institute of Chemical Technology, Hyderabad for

support and providing the necessary facilities and also thanks to Director General, CCRUM,

New Delhi for giving constant encouragement and support. The author also thank Project

Director, NIPER Hyderabad for providing the LCMS facility.

Conflict of interest: None

REFERENCES

1. The Unani Pharmacopoeia of India Part- I, Volume-I, Department of AYUSH, Ministry

of Health and Family Welfare, Government of India, p. 48.

2. Tanaka T, Linuma M, Fuji Y, Yuki K, Mizuno M. Flavonoids in root and bark of

Pongamia pinnata. Phytochemistry, 1992; 31: 993- 998.

3. Simin K, Ali Z, Khaliq-Uz-Zaman SM, Ahmad VU. Structure and biological activity of a

new rotenoid from Pongamia pinnata, Natural Product Letters, 2002; 16(5): 351-357.


856


4. Pavanaram SK, Ramachandra Row L. New flavones from Pongamia pinnata (L.) Merr.:

identification of compound D. Nature, 1955; 176: 1177.

5. Aneja R, Khanna RN, Seshadri TR. 6-Methoxyfuroflavone, a new component of the

seeds of Pongamia glabra. Journal of the Chemical Society, 1963; p.163-168.

6. Warrier PK, Nambiar VPK, Ramakutty C. Indian Medicinal Plants. Vol. IV. Orient

Longman Ltd., Madras, 1995; pp. 339.

7. Singh RK, Pandey BL. Anti-inflammatory activity of seed extracts of Pongamia pinnata

in rats. Indian Journal of Physiology and Pharmacology, 1996; 40: 355-358.

8. Dahanukumar SA, Kulkarni RA, Rege NN. Pharmacology of medicinal plants and natural

products. Indian Journal of pharmacology, 2000; 32: 81-118.

9. Prasad G, Reshmi MV. A manual of medicinal trees. Agrobios India. Propagation

methods. Foundation for revitalization for local health tradition, India, 2003; pp.132.

10. Sridhar NB. Effect of dermovet ointment in treating dermatitis of domestic animals.

Indian Journal of Animal Research, 2009; 43(3): 211-212.

11. Shejawal N, Menon S, Shailajan S. Bioavailability of karanjin from Pongamia pinnata L.

in sprague dawley rats using validated RP-HPLC method. Journal of Applied

Pharmaceutical Science, 2014; 4(3): 010-014.

12. Dharmesh SM, Vismaya, Belagihally SM, Rajashekhar S, Jayaram VB, Kanya S, et al.,

Gastroprotective properties of karanjin from karanja (Pongamia pinnata) seeds; role as

antioxidant and H+, K+-ATpase inhibitor. Evidence-Based Complementary and

Alternative Medicine, 2011; 20: 1-10.

13. Vismaya, Eipeson WS, Manjunatha JR, Srinivas P, Kanyaa TCS. Extraction and recovery

of karanjin: A value addition to karanja (Pongamia pinnata) seed oil. Industrial Crops and

Products, 2010; 32: 118–122.

14. Yadav RD,. Jain SK, Alok S, Prajapati SK, Verma A. Pongamia Pinnata: An overview.

International Journal of Pharmaceutical Sciences and Research, 2011; 2(3): 494-500.

15. Lanjhiyana S, Patra KC, Ahirwar D, Rana AC, Garabadu D, Lanjhiyana SK. Karanjin in

Pongamia pinnata: A novel Indian medicinal plant. Der Pharmacia Sinica, 2012; 3(1):

144-147.

16. Tamrakar AK, Yadav PP, Tiwari P, Maurya R, Srivastava AK. Identification of

pongamol and karanjin as lead compounds with antihyperglycemic activity from

Pongamia pinnata fruits. Journal of Ethnopharmacology, 2008; 118(3): 435-9.


857


17. Patel PP, Trivedi ND. Simple, efficient and economic method for isolation and analysis of

karanjin and pongamol from karanja seed oil and screening of antimicrobial potential.

International Journal of Pharmacy and Pharmaceutical Sciences, 2015; 7(7): 248-252.

18. Parmar BS, Gulati KC. Synergists for pyrethrins (II)-karanjin. Indian Journal of

Entomology, 1969; 31: 239–243.

19. Shailajan S, Menon S, Pednekar S, Singh A. Wound healing efficacy of Jatyadi Taila: in

vivo evaluation in rat using excision wound model. Journal of Ethnopharmacology, 2011;

138(1): 99-104.

20. Chopade VV, Tankar AN, Pande VV, Tekade AR, Gowekar NM, Bhandari SR,

Khandake SN. International Journal of Green Pharmacy, 2010; 10: 72-75.

21. Gore VK, Satyamoorthy P. Determination of pongamol and karanjin in karanja oil by

reverse phase HPLC. Analytical Letters, 2000; 33(2): 337-346.

22. Guideline on validation of analytical procedure-methodology. International Conference

on Harmonization, Geneva, Switzerland, 1996.

World Journal of Pharmaceutical Research et al …KEYWORDS: Karanj oil, Pongamia Pinnata, UPLC,...

Documents

Transcript of World Journal of Pharmaceutical Research et al …KEYWORDS: Karanj oil, Pongamia Pinnata, UPLC,...